PROJECT SUMMARY (High-resolution and haplotype-specific mapping technology of 3D genome organization) It is well known that the three-dimensional (3D) organization of genomic DNA is mediated by protein factors within the nuclear space. Such 3D organization is also speculated to be affected by the presence of abundant non-coding RNAs (ncRNAs), particularly long ncRNAs (lncRNAs). Thus, 3D mapping of this complex system including 46 chromosomes of the human genome and interactions with many proteins and lncRNAs (nucleome) requires the development of robust methods with high resolution and haplotype specificity in genome-wide manner. Our ultimate goal is to deliver a nucleome positioning system (NPS) composed of complex chromatin interaction network maps in the context of 3D genome structures, from which the dynamics of individual genomic elements can be monitored and referenced. To achieve this goal, we plan to develop a set of high throughput 3D mapping technologies that will be used to generate multi-scale, high-resolution and haplotype-specific chromatin interaction data to model the ensemble structures of spatiotemporal organization of the human genome. Among all existing 3D mapping technologies, ChIA-PET and RICh-PET have been demonstrated to have this potential, but still require further refinements to increase efficiency and reduce the input cell sample, so as to enhance their suitability across a broader spectrum of cell types and biological questions. In this Mapping Technology Development Component, we propose a series of sophisticated, integrated approaches to significantly improve the ChIA-PET and RICh-PET protocols. Specifically, we will i) Improve experimental efficiency in key steps in the ChIA-PET/RICh-PET protocols, namely by adapting Tn5 transposase-mediated tagmentation and a new annealing and extension (A&E) step to replace inefficient DNA cutting/ligation steps and to streamline the entire protocol, which will also increase tag-read length and improve tag-mapping accuracy and coverage (Aim 1); and ii) adapt a microfluidics-based platform to enable process miniaturization and streamline automation of the sample preparation workflow (Aim 2). Successful completion of these technology advancements will significantly enhance the robustness of ChIA-PET and RICh-PET for the generation of high-resolution, high-quality ensemble 3D maps of human genomes, will enhance the applicability of these powerful technologies to a broader range of cell types, including those of low input quantity, and will set the standard for future studies of chromatin interactions.